PROJECT SUMMARY/ABSTRACT Multiple myeloma is an aggressive hematologic malignancy that remains incurable despite recent progress. This disease of malignant plasma cells is fundamentally associated with aberrant protein homeostasis, defined by an extremely high burden of immunoglobulin synthesis. Proteasome inhibitors (PIs), a widely-used first-line therapy in myeloma, are thought to directly take advantage of this aberrancy by increasing unfolded protein stress leading to cell death. However, this mechanism is not fully proven, and further insight into PI-induced cell death may lead to more effective combination strategies. In addition, PI resistance is a major clinical problem in myeloma, and new strategies are needed to overcome this condition. Here, we hypothesize that the remodeling of the plasma cell proteome after therapy is central to both PI response and resistance. We specifically propose that proteome remodeling is mediated through rewiring of proteostasis pathways involving chaperones, the VCP/p97 complex, and the ubiquitin-proteasome system, as well as through changes to the alternative splicing landscape, as mediated by post-translational modification of the splicing machinery. To explore this hypothesis we will take advantage of novel pharmacologic and genetic perturbation tools, cellular and biochemical assays, in vivo models, clinical trial genomic data, primary sample analysis, RNA sequencing, and mass spectrometry approaches. The overall goals of this proposal are 1) develop new therapy strategies either in combination with PIs or in the PI-refractory setting, and 2) describe a new, systematic approach to probe the architecture of proteostasis networks. Importantly, our preliminary results challenge existing paradigms related to PI efficacy. In Aim 1, we address paradoxical findings relating the unfolded protein response, the interaction between the p97 degradation machinery and PIs, and the relevance of inducible HSP-family chaperones. We will take advantage of novel pharmacology available to us, including active site and allosteric inhibitors of p97 and allosteric inhibitors of HSP70, in combination with functional genetics by CRISPR interference, to define the role of central protein homeostasis nodes defining PI response and resistance. Furthermore, we will use our unique expertise in pulsed-SILAC proteomics to determine specific substrates of the p97 machinery and the proteasome in the presence of clinically-relevant resistance modifications. Toward Aim 2, our preliminary studies using unbiased mass spectrometry have revealed significant phosphorylation of the spliceosome after PI treatment. We first aim to characterize the relationship between specific alternative splicing events and proteome remodeling after PIs. We then aim to extend our promising preliminary data demonstrating the efficacy of splicing inhibitors as a new anti-myeloma therapy. Overall, the studies here will have a direct impact on delineating the surprisingly broad range of PI-mediated effects in plasma cells, validate the novel therapeutic strategy of splicing inhibition, and reveal new mechanistic approaches to dissect proteostasis networks and alternative splicing that could extend far beyond myeloma.